Sense of belonging and involvement has shown to be positively associated with academic achievement and retention. We argue that a focus on sense of belonging and involvement specifically at the departmental level is valuable, yet rarely discussed in the literature. In this article, we describe the development and evaluation of a questionnaire measuring university students' sense of belonging to and involvement in their "home" department (biology). The questionnaire was named the DeSBI questionnaire (Departmental Sense of Belonging and Involvement) and was administered to students attending an R1 university in the southeastern United States during Spring 2018 (n = 201) and Fall 2018 (n = 737) semesters. Factor analysis indicated a three-factor solution: one factor consisting of five items representing sense of belonging: valued competence; a second factor consisting of six items representing sense of belonging: social acceptance; and a third factor consisting of nine involvement items. Analysis of variance showed significant differences in all subscales between students included in a special biology program, biology majors, and non-biology majors. The study findings support the use of the instrument for measuring biology students' sense of belonging to and involvement in their biology department.